The ability of pluripotent stem cells to generate various replacement cells and tissues highlights their potential application in cell-based regenerative therapies. Although human embryonic stem cells (ESCs) and inducted pluripotent stem cells (iPS) are versatile sources of stem cells with multiple differentiation potential, however concerns such as teratoma formation and immunogenic rejection remain as obstacles. Pluripotent stem cells reside in adult human tissues derived from neural crest (NC) and may participate in tissue regeneration or repair.This suggest that the adult patients' own pluripotent stem cells could be isolated for the treatment of diseases, thus avoiding the potential problems of ESC transplantation. During development, NC originating at the dorsal region of the neural tube contains pluripotent cells that contribute to development of varieties of organs/tissues in the body. Periodontal ligament (PDL) is derived from cranial NC. Our recent study showed that cells isolated from adult human PDL expressed ESC markers ( Oct4, Nanog, Sox2) and NC markers (p75, Nestin, Sox10), and exhibited the potential to differentiate into all 3 germ layers. This suggests that PDL stem cells with ESC or NC characteristics may indeed exhibit pluripotency. The value and significance of discovering cell populations within adult tissues that possess an ESC-like phenotype and pluripotent behavior cannot be overstated. Cigarette smoking is a well-known risk factor for varieties of diseases. Significant tissue hypoxia mainly attributed to carbon monoxide (CO) and nicotine. CO raises carboxyl- hemoglobin levels, whilst nicotine induces vasoconstriction, which further decreases tissue oxygenation. Smoking delays skeletal healing by 60% following fractures. Nicotine delays bone growth by influencing gene expression in bone healing process. It is unclear whether cigarette smoking affects endogenous stem cells. Since oral tissues are first to come in direct contact with smoking, this is a unique opportunity to examine the effect of smoking on the viability and regenerative/differentiation potential of this unique population of pluripotent stem cell population. Our hypothesis is that a homogeneous population of pluripotent stem cells can be isolated from human adult periodontal ligament. Tissue hypoxia caused by CO and nicotine from smoking greatly reduce regenerative potential and viability of this population of stem cells. We have 3 aims. Specific Aim 1: To isolate and further characterize a homogeneous population of pluripotent stem cells from adult Periodontal Ligament. Based on our institutional strengths on cellular therapies on cardiovascular diseases, cartilage and bone repair, Specific Aim 2a: We shall compare the viability of stem cells and their regenerative potential e.g. osteogenesis isolated form PDL from the age-match smokers to nonsmokers. Specific Aim 2b: Evaluate the effect of smoking on the subpopulation of PDL cells that may be important for cardiomyogenesis. Specific Aim 2c: To evaluate the mechanism of cardiomyogenic differentiation of PDLSC to mechanical induction and utilize this response in the enhancement of current differentiation protocols. Specific Aim 3: To evaluate the in vivo engraftment potential of the pluripotent human PDLSC.